In scanning microscopy, a sample is illuminated with a light beam in order to observe the detected light, constituting reflected or fluorescent light, emitted by the sample. The focus of an illuminating light beam is moved in a sample plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detected light coming from the specimen is measured as a function of the position of the scanning beam, and the detected values thus ascertained are allocated to position values. In order to ascertain the position values, the positioning elements are usually equipped with sensors that ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an diaphragm (called the “excitation diaphragm), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection diaphragm, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels by way of the beam deflection device back to the beam splitter, passes through it, and is then focused onto the detection diaphragm behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection diaphragm, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
German Patent Application DE 198 29 944 A1 discloses a method for device configuration, preferably of laser scanning microscopes, in which laser light having one or more spectral lines is generated and is directed onto a sample which contains a fluorescent dye or onto which a fluorescent dye is applied. The excitation wavelengths and emission wavelengths of different fluorescent dyes are acquired in separate data sets and are stored in a data memory. The laser spectra that can be set with the microscope and are to be directed onto the sample, and the transmission spectra achievable with the available filters, are also acquired in data sets and those data sets are saved. Parameters for configuration of the microscope are ascertained from a computational combination of said data sets. The device configuration concerns the selection of the laser line of the excitation laser, and the selection of suitable filters.
German Patent Application DE 43 30 347 A1 discloses an apparatus for the selection and detection of at least two spectral regions of a light beam, having a selection device and a detection device. For reliable simultaneous selection and detection of different spectral regions at high yield and with the simplest possible design, the apparatus is configured in such a way that the selection device comprises means for spectral dispersion of the light beam and means on the one hand for blocking out a first spectral region and on the other hand for reflecting at least a portion of the unblocked spectral region, and the detection device comprises a first detector arranged in the beam path of the blocked-out first spectral region and a second detector arranged in the beam path of the reflected spectral region. A slit diaphragm arrangement having mirror-coated diaphragm panels is provided as the means for blocking out a first spectral region and on the other hand for reflecting at least a portion of the unblocked spectral region. The apparatus is usable in particular as a multi-band detector in a scanning microscope.
The known scanning microscopes and known methods have the disadvantage that a largely accurate quantitative spectral analysis of the light proceeding from a sample is not possible.